RESEARCH
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Open Access
Antifungal activity, mechanical properties, and accuracy of three-dimensionally printed denture base with microencapsulated
phytochemicals on varying post-polymerization time
Ye‑Hyeon Jo1†, Won‑Jun Lee2†, Ji‑Hyun Lee1 and Hyung‑In Yoon2*
Abstract
Background: Studies on the antifungal activity, flexural strength, Vickers hardness, and intaglio surface trueness of three‑dimensionally printed (3DP) denture bases with microencapsulated phytochemicals with respect to changes in post‑polymerization time (PPT) are lacking.
Methods: Specimens of various shapes and dimensions were fabricated with a 3DP denture base resin mixed with 5 wt% phytoncide‑filled microcapsules. Each specimen was subjected to different PPT protocols of 5, 10, 20, and 30 min. Specimens without microcapsules with 5‑min PPT were used as the negative control group. Cell colonies were counted to evaluate antifungal activity. Three‑point bending and Vickers hardness tests were performed to measure the flexural strengths and hardness of the specimens. Fourier‑transform infrared spectrometry was used to inspect the degree of conversion (DC). The intaglio surface trueness was measured using root‑mean‑square estimates calculated by superimposition analysis. A non‑parametric Kruskal–Wallis test or one‑way analysis of variance was performed (α = 0.05).
Results: The specimens with microcapsules and 10‑min PPT showed the highest antifungal activity among the tested groups. Compared with the positive control group (5‑min PPT), the specimens with PPTs of 10 min or longer showed significantly higher mean flexural strength, higher DC, greater hardness, and better trueness (all, P < 0.05).
Except for the difference in antifungal activity, no statistically significant differences were detected between the speci‑
mens subjected to 10‑, 20‑, and 30‑min PPT.
Conclusion: The 3DP denture base filled with microencapsulated phytoncide showed different antifungal activity and physical properties on changing PPT. The 3DP denture base containing phytoncide‑filled microcapsules at 5 wt%
†Ye‑Hyeon Jo and Won‑Jun Lee contributed equally to this work as first authors.
*Correspondence: drhiy226@snu.ac.kr
2 Department of Prosthodontics, School of Dentistry and Dental Research Institute, Seoul National University, 101, Daehak‑ro, Jongno‑gu, Seoul 03080, Republic of Korea
Full list of author information is available at the end of the article
concentration and subjected to 10‑min PPT exhibited sufficient antifungal activity as well as mechanical properties and accuracy within clinical acceptance.
Keywords: 3D printing, Denture base, Phytoncide, Microcapsule, Antifungal activity, Post‑polymerization time
Introduction
Polymethyl methacrylate (PMMA) resin has been used in the fabrication of removable denture prostheses for a long time [1–3]. It has several advantages, such as favorable esthetics, biocompatibility, low cost, and simple process- ing [1, 4]. However, fracture of the acrylic resin is fre- quently reported in 57–64% of all cases of clinical failures of removable denture prostheses [5, 6]. Furthermore, the conventional pack and press technique of denture fabri- cation has been reported to be susceptible to dimensional change owing to polymerization shrinkage [7, 8]. With the progress of computer-aided design and computer- aided manufacturing (CAD-CAM) technology, additive manufacturing methods, also known as three-dimen- sional (3D) printing, have been applied to the workflow of fabricating denture prostheses [9, 10]. 3D-printed (3DP) denture bases can be manufactured with unlimited shape reproduction, sufficient precision, and physical properties using a photo-polymerizable acrylic resin [9, 10], which simplifies the workflow of removable dentures [10]. Recently, the clinical applicability of 3DP denture bases has been substantiated by several researchers. Den- ture bases for edentulous arches fabricated using digital light processing (DLP) showed favorable tissue surface adaptation and dimensional accuracy, which was similar to or better than that of bases manufactured by 5-axis milling [11, 12]. A clinical study by Yoon et al. revealed that denture bases produced via 3D printing were more advantageous than those produced via milling in terms of tissue surface adaptation due to better reproduction of the complex contour of the edentulous ridge [13]. Fur- thermore, the adaptation of the denture base to the tissue surface can be improved by optimizing the build angle during the DLP-based workflow [14]. However, regard- less of the manufacturing workflow, the acrylic resin material used for denture bases has an inherent draw- back, as the intaglio surface could be a potential reservoir of oral microorganisms, such as bacteria or fungi, due to surface roughness, geometric irregularities, and porosity [15–17].
Denture stomatitis is an inflammatory fungal infec- tion that affects approximately 50–75% of otherwise healthy denture wearers and is the most common form of oral candidiasis [18–20]. Apart from antifungal treat- ment, conditioning of the abused tissue, cleansing of the affected mucosa or denture surface, and fabrica- tion of new dentures are the treatment of choice. Due to
concerns regarding systemic adverse reactions caused by oral antifungal drugs [21, 22], alternative approaches, such as photodynamic treatment [23] or adjunct physi- cal cleansing, have been recently used to effectively treat denture stomatitis [24]. In addition, imparting antibacte- rial activity to the denture material by directly coating or adding antibacterial compounds has also been proposed [25–28]. In particular, a novel additive manufacturing technique to directly add substances with antibacterial properties to the denture material has been suggested.
The substances selected for this purpose were certain oxides or chemical compounds and phytochemicals, which are naturally derived substances with antimicro- bial activities [29–31]. The antifungal properties of phy- tochemicals, such as phytoncide oil, have been previously reported [32, 33]. The phytoncide could reduce optical density and viability of fungal cells as well as increase the number of morphologically atypical cells [34]. Recent studies reported the development of a denture base with antifungal properties by incorporating phytoncide into PMMA resin [35, 36]. Furthermore, microencapsulation of phytoncide oil extract was recommended to achieve continuous effect of its antimicrobial activity [30, 36].
A novel method using microencapsulation and additive manufacturing has been recently introduced to incor- porate the antifungal activity of phytoncide extract into the acrylic resin for 3DP denture bases and maintain its effectiveness for a certain period without compromise [31]. 3DP denture bases with phytoncide-filled microcap- sules at concentrations ranging from 6 to 8 wt% showed sufficient antifungal effects on fungal cell colonies [31].
However, they showed mediocre mechanical proper- ties (flexural strength or hardness), which were barely comparable with those of bases without microcapsules, and exhibited lesser dimensional accuracy, which was clinically acceptable [37]. Since denture bases should withstand masticatory forces, resist surface wear, and guarantee intimate tissue surface adaptation, the physi- cal properties and dimensional accuracy of the 3DP den- ture base with phytoncide-filled microcapsules must be improved further. Among the various parameters used for 3D printing, the light exposure time during the post- polymerization has been reported to affect the accuracy and strength of 3DP resin materials [38–40]. The dimen- sional accuracy (trueness and precision), degree of con- version, and mechanical properties (flexural strength and Vickers hardness) of resin-based 3DP objects have been
significantly improved by changing the post-polymeriza- tion time (PPT) [38–40].
To the best of our knowledge, there has been no research on the effect of PPT on the antifungal activity, physical properties, and accuracy of 3DP denture base resins containing phytochemical-filled microcapsules.
Therefore, this in-vitro study aimed to evaluate 3DP den- ture bases with microencapsulated phytochemical (phyt- oncide oil extract) in terms of antifungal activity, flexural strength, Vickers hardness, and intaglio surface trueness with respect to changes in PPT (5, 10, 20, and 30 min).
The null hypothesis was that the antifungal activity, flex- ural strength, Vickers hardness, and intaglio surface true- ness of 3DP denture bases filled with microencapsulated phytoncide would not be affected by the changes in PPT.
Methods
A disc-shaped specimen with a diameter of 15 mm and thickness of 5 mm was virtually designed with uni- versal CAD software (TinkerCAD, Autodesk, Que- bec) to test the antifungal activity, Vickers hardness, and degree of conversion. A bar-shaped specimen (65 × 10 × 3.3 ± 0.2 mm) was designed to measure the flexural strength of the printed denture base materials in accordance with ISO 20975-1:2013 [41]. A maxillary denture base-shaped specimen was designed to evalu- ate the trueness of the intaglio surface after 3D print- ing. An edentulous maxillary dentiform with soft gingiva (EDE1001-UL-UP-DPM, Nissin, Japan) was digitized to obtain maxillary full-arch scan data (i500; Medit, Korea).
A maxillary complete denture base (reduced to 50% of its size) was virtually designed using dental CAD software (3Shape Dental Designer; 3Shape, Danmark) and saved in a standard tessellation language (.stl) format as refer- ence CAD data.
A 3D-printable resin (NextDent Denture 3D+, Vertex Dental BV, Soesterberg, Netherlands), designed for the fabrication of removable denture bases, was used in this study. Phytoncide oil was extracted from Pinus Densi- flora (Cleandiox. Ltd., Korea) and microencapsulated to prepare functional microcapsules with antifungal activity [31]. Detailed process of microencapsulation was previ- ously reported in the study by Jeon et al. [31]. Briefly, the phytoncide oil and the styrene-maleic anhydride copoly- mer solution was mixed to form droplets. Melamine/
urea/formaldehyde was then dissolved in the mixture to form a wall of the microcapsule. After aging for cohesion, the microcapsules were sieved and dried. The phyton- cide-filled microcapsules were carefully mixed with the 3D-printable denture base resin at a concentration of 5 wt% [31, 37]. A dispersant (DISPERBYK-111, BYK, Ger- many) was added at 20 wt% of the mass (g) of the phy- toncide-filled microcapsules to the mixture for uniform
dispersion. All 3DP denture base resin specimens with various shapes and dimensions were produced using the microcapsule-resin mixture and a DLP-based 3D printer (MAX UV, ASIGA, Australia) with a wavelength of 385 nm. The layer thickness and build orientation for each 3D printing were set to 50 μm and 0°, respec- tively, and horizontally positioned to build the platform (Asiga Composer 1.2.12, Asiga, Australia). Specimens of 3DP denture base resin with no microcapsules and dis- persant agent were also manufactured according to the abovementioned protocols and parameters as the con- trol group. All specimens were cleaned using ethanol in a washing machine as per the manufacturer’s instructions (Form wash, Formlabs, Somerville, USA) for 10 min. The support structures were carefully removed, and no fur- ther adjustments were made to the specimen surfaces.
After cleansing and removing the support structure, the disc-shaped 3DP denture resin specimens (Fig. 1) underwent post-polymerization with a curing unit (Cure M U102H, Graphy, Seoul, Korea). The 5-min PPT, a default protocol recommended by the manufacturer, was applied to the 3DP denture resin discs with phytoncide- filled microcapsules for the control group (positive con- trol). For the experimental groups, 3DP denture resin discs with microcapsules subjected to three different PPTs (10-, 20-, and 30-min) were evaluated. As a nega- tive control, the 3DP denture resin discs without micro- capsules subjected to 5-min PPT (default protocol) were also tested. After post-processing as recommended by the manufacturers, the as-printed surface of each 3DP denture resin specimen, opposing the attached area of the support structures, was used as a testing surface for further analysis.
Candida albicans (ATCC 10,231, C. albicans), pro- vided by the Korean Collection for Oral Microorgan- isms, was cultured in Sabouraud dextrose (SD) broth at 37 °C and used to assess the antifungal activity of the 3DP denture resin discs with phytoncide-filled micro- capsules. The 3DP discs with and without microcapsules (n = 5 each), subjected to different PPT protocols, were placed at the bottom of a 12-well plate. To each well, 2 mL of C. albicans suspension (optical density: 0.02, equivalent to 2 × 104 microbial cells per mL) was added and incubated for 24 h at 37 °C. The discs were rinsed with Dulbecco’s phosphate-buffered saline (DPBS) and transferred to tubes filled with 5 mL of SD broth. Each disc was ultrasonicated at 40 kHz for 5 min to detach the microbial cells from its surface (NXPC-B5020SB, Kodo, Korea) [42]. The absorbance value of the fungal solu- tion (200 µL) was measured using a microplate reader (Epoch2, BioTek, USA) at a wavelength of 600 nm.
Subsequently, 10 µL of the diluted fungal solution was seeded onto an SD agar plate and incubated for 24 h at
37 °C. For the SEM analysis, the discs were cleaned with DPBS and fixed in 2.5% (v/v) glutaraldehyde (Sigma- Aldrich, USA) for 2 h. The fixative was aspirated and cleaned with DPBS and post-fixed in 1% osmium tetrox- ide for 30 min. After fixation, samples were dehydrated in a graded series of ethanol solutions. After critical point drying, the specimens were sputter-coated with Pt and microscopically examined using field-emission scan- ning electron microscopy (FE-SEM, Apreo S, Thermo Fisher Scientific, MA, USA) at 10 kV. The colonies were counted as colony-forming units per milliliter (CFU/
mL), and the morphological changes in the fungal cell colonies on the specimen surfaces were examined.
To measure the flexural strength of the denture base materials in accordance with ISO 20795-1:2013 [41], the bar-shaped 3DP denture resin specimens (n = 10) sub- jected to different PPTs with and without microcapsules were prepared and stored in 37 °C distilled water for 50 h.
The flexural strength of each bar-shaped specimen was measured using a three-point bending test apparatus with a universal testing machine (Instron 8871; Instron, Canton, MA, USA) of 5kN load cell at a crosshead speed of 5 mm/min. The flexural strength (FS) was computed using the following equation:
where P is the maximum load before fracture, l is the distance between the supports (50 mm), b is the speci- men width, and d is the specimen thickness. After test- ing, the fractured surfaces were sputter-coated with Pt (5-nm layer thickness) and observed using FE-SEM for fractographic analysis.
A Fourier transform infrared spectrometer (FT-IR, TENSOR27, Bruker, Germany) and data collection pro- gram (OPUS, OPtical User Software, Bruker, Germany) was used to investigate the effect of PPT on the degree of conversion (DC) of the 3DP denture resin specimens with microcapsules. Considering the PPT period, the infrared spectra of 3DP denture resin discs containing phytoncide- filled microcapsules, as well as those without microcap- sules, were recorded between 4000 cm− 1 and 400 cm− 1 in the absorbance mode (n = 3 per group), with a reso- lution of 1 cm− 1. The FTIR spectra of unpolymerized denture base resins were also recorded. DC of each 3DP denture resin specimen was calculated using the following equation.
FS = 3Pl 2bd2,
Fig. 1 Colony‑forming units per milliliter (CFU/mL) of Candida albicans detached from 3D‑printed denture base resin discs and subjected to different post‑polymerization time (PPT) protocols: 5‑min, 10‑min, 20‑min, and 30‑min. Control: 3D‑printed denture base resin discs without microcapsules; Capsule: 3D‑printed denture base resin discs with 5 wt% phytoncide‑filled microcapsules. Significant differences between the groups are marked with black lines (P < 0.05)
where Absaliphatic is the absorbance peak intensity of the aliphatic bond of the 3DP denture resin material meas- ured from the peak at 1638 cm− 1 and Absaromatic is the absorbance peak intensity of the aromatic bond of the 3DP denture resin material measured from the peak at 1610 cm− 1.
To investigate the hardness of the 3DP denture resin disc, the discs with and without phytoncide microcap- sules, subjected to different PPT protocols (n = 3 per group), were prepared. Minimizing the effect of micro- capsules on the surface topography, the testing surface of each disc was polished for clear examination of inden- tation using a polishing machine (Phoenix Beta, Bühler, Düsseldorf, Germany) at 150 rpm with 4000 grit SiC papers. The Vickers hardness test was performed on the polished surface of each disc using a diamond pyramid indenter (HM-220B, Mitutoyo, Japan) at 9.8 N load and repeated 10 times for each disc-shaped specimen.
The 3DP denture base-shaped specimens with and without microcapsules were subjected to different PPT protocols (n = 10 per group), and their intaglio surfaces were digitized using a high-resolution laboratory scan- ner (Medit T710; Medit Corp, Seoul, Korea) in standard operation mode. The intaglio surface trueness of each specimen was evaluated using a 3D inspection software (Geomagic Control X; 3D Systems, Rock Hill, SC, USA) by superimposing the intaglio area of the reference CAD data of the denture base and the scanned intaglio sur- face data of the specimens. Three pairs of correspond- ing points were carefully selected on the tissue surface of each scanned denture base and reference CAD data to achieve preliminary alignment. Subsequently, the best- fit alignment with the iterative closest point-matching algorithm was performed. A color-coded 3D deviation map was displayed for each superimposition analysis of the 3DP specimens. The nominal deviation was set to
± 50 μm, and the critical deviation was set to ± 500 μm.
The area within nominal deviation was displayed in green, while the area greater than nominal deviation was displayed in yellow (positive) or blue (negative). The surface deviation data measured in root-mean-square estimates (RMS, µm) were calculated using the formula below:
Degree of Conversion(DC) =
1 − Absaliphatic polymer /Absaromatic polymer
Absaliphatic(monomer)/Absaromatic(monomer)
×100
RMS =
n
i=1 X1,i− X2,i 2
√n
where X1,i is the point i on the reference data; X2,i is the point i on the test data; n is the total number of points.
The statistical analyses were conducted using the measured data of the 3DP denture resin specimens from five different treatment categories: (1) no microcap- sule and 5-min PPT (negative control), (2) microcapsule and 5-min PPT (positive control), (3) microcapsule and 10-min PPT, (4) microcapsule and 20-min PPT, and (5) microcapsule and 30-min PPT. Based on the distribution of the collected data tested by the Shapiro–Wilk’s method and Levene’s test of equal variance, a non-parametric Kruskal–Wallis test and a post-hoc multiple comparison with Wilcoxon rank test adjusted by Bonferroni’s method were conducted for the data collected from the antifungal activity and degree of conversion analyses. For the meas- urements of each test for flexural strength (MPa), Vickers hardness (VH), and intaglio surface trueness (RMS, µm), one-way analysis of variance (ANOVA) and a post-hoc multiple comparison using Tukey’s method were per- formed for the differences among the groups. All statisti- cal analyses were performed using IBM SPSS Statistics, v25.0; IBM Corp., Armonk, NY, USA), with a level of sta- tistical significance (α) of 0.05.
Results
Based on the Kruskal-Wallis test, the numbers of fungal cell colonies (CFU/mL) on the discs with microencap- sulated phytoncide were significantly different between the groups (Fig. 1, P < 0.001). Adherence of fungal cell colonies to the disc surface was the lowest in the speci- mens with microcapsules and 10-min PPT, which indi- cates the highest antifungal activity. The specimens with microcapsules and 5-min PPT (positive control) showed significantly higher antifungal activity than those with- out microcapsules and 5-min PPT (negative control, P < 0.05). However, as the PPT increases from 10 to 30 min, the numbers of fungal cell colonies attached to the discs filled with microcapsules significantly increased (all P < 0.05).
Figure 2 shows the mean flexural strength values of the specimens subjected to different PPT protocols.
The mean strength value of the specimens with no microcapsules and 5-min PPT (negative control) was 72.45 ± 10.46 MPa. With the same PPT, incorporat- ing microcapsules into the denture base resin signifi- cantly decreased the mean value to 52.35 ± 10.26 MPa (positive control, P < 0.05). However, when subjected
to 10-min PPT, the mean strength value of the speci- mens with microcapsules significantly increased up to 67.09 ± 4.47 MPa (P < 0.05). As the PPT increased to 20 and 30 min, the specimens with microcapsules showed significantly improved mean strength values compared with those from the positive control group (all, P < 0.05).
The specimen with microcapsules and treated with 10-min PPT revealed comparable strength as those with- out microcapsules (negative control, P > 0.05). Micro- scopic observations (Fig. 3) revealed that the specimens from the negative control group showed brittle fracture patterns with flat, compact, and organized surfaces.
Fig. 2 Flexural strength values (MPa) of bar‑shaped 3D‑printed denture base resin specimens subjected to different post‑polymerization time (PPT) protocols: 5‑min, 10‑min, 20‑min, and 30‑min. Control: 3D‑printed denture base resin specimens without microcapsules; Capsule: 3D‑printed denture base resin specimens with 5 wt% phytoncide‑filled microcapsules. Significant differences between the groups are marked with black lines (P < 0.05). Flexural strength value of 65 MPa is marked in red dotted line
Fig. 3 Representative microscopic images (upper row, ×200, and lower row, ×2000) of fractured 3D‑printed bar‑shaped denture base specimens subjected to different post‑polymerization time (PPT) protocols: 5‑min, 10‑min, 20‑min, and 30‑min. Control: 3D‑printed denture base resin discs without microcapsules; Capsule: 3D‑printed denture base resin discs with 5 wt% phytoncide‑filled microcapsules. a Brittle fracture with smooth surface and chevron marks; b–e intermediate (ductile) fracture with rough surface and jagged areas. Note the microcapsules are evenly distributed over the fractured surfaces
However, irrespective of the PPT protocols, all specimens with microcapsules showed typical patterns of interme- diate fractures.
The DC values of the discs with microencapsu- lated phytoncide were significantly different between the groups, according to the Kruskal-Wallis analy- sis (P = 0.014). The 3DP denture resin discs filled with microcapsules and subjected to PPT for no less than 10 min showed significantly improved DC compared with those subjected to 5-min PPT (P < 0.05, Fig. 4). From the highest to lowest of the DC values, the discs subjected to 20-min PPT (52.01 ± 0.05%) were followed by those subjected to 10-min PPT (50.68 ± 0.77%), 30-min PPT (48.31 ± 1.51%), and 5-min PPT (34.62 ± 3.32%, positive control). No difference was found between the DC values of the specimens from the positive and negative control groups (P > 0.05). In addition, there was no statistical dif- ference in the DC values between the specimens treated with 10-, 20-, and 30-min PPTs (all, P > 0.05).
The mean hardness value (HV) of the specimens with- out microcapsules and subjected to 5-min PPT (negative control) was significantly higher than that of those with microcapsules, regardless of the changes in PPT proto- cols (all, P < 0.05, Fig. 5). The mean hardness value of the specimens from the positive control group (18.06 ± 0.37)
was the lowest among the tested groups. However, on using PPT protocols of no less than 10 min, the mean hardness values of the specimens with microcapsules significantly increased compared with that of those subjected to 5-min PPT (all, P < 0.05). The mean hard- ness value was 18.99 ± 0.36 for the specimens subjected to 10-min PPT. No difference was detected between the groups filled with microcapsules and subjected to 10-min, 20-min, and 30-min PPT (all, P > 0.05).
The highest mean RMS value was 39.30 ± 4.00 μm for the specimens with microcapsules and sub- jected to 5-min PPT. In contrast, the lowest value was 29.73 ± 4.60 μm for those subjected to 20-min PPT.
Under the same PPT of 5 min, the specimens with microcapsules showed significantly inferior true- ness of the intaglio surface compared with that of those without microcapsules (P < 0.05, Fig. 6). As the PPT increased, all specimens with microcapsules and subjected to PPT for no less than 10 min showed better trueness than those subjected to 5-min PPT (positive control, P < 0.05). No statistical differences were detected between the specimens with microcap- sules and subjected to 10-min, 20-min, and 30-min PPT protocols (all, P > 0.05). Furthermore, the speci- mens filled with microcapsules and treated with PPT Fig. 4 Degree of conversion (%) of 3D‑printed denture base resin discs subjected to different post‑polymerization time (PPT) protocols: 5‑min, 10‑min, 20‑min, and 30‑min. Control: 3D‑printed denture base resin discs without microcapsules; Capsule: 3D‑printed denture base resin discs with 5 wt% phytoncide‑filled microcapsules. Significant differences between the groups are marked with black lines (P < 0.05)
Fig. 5 Vickers hardness (VH) of 3D‑printed denture base resin discs subjected to different post‑polymerization time (PPT) protocols: 5‑min, 10‑min, 20‑min, and 30‑min. Control: 3D‑printed denture base resin discs without microcapsules; Capsule: 3D‑printed denture base resin discs with 5 wt%
phytoncide‑filled microcapsules. Significant differences between the groups are marked with black lines (P < 0.05)
Fig. 6 Root‑mean‑square estimates of the 3D‑printed denture base resin discs subjected to different post‑polymerization time (PPT) protocols:
5‑min, 10‑min, 20‑min, and 30‑min. Control: 3D‑printed denture base resin discs without microcapsules; Capsule: 3D‑printed denture base resin discs with 5 wt% phytoncide‑filled microcapsules. Significant differences between the groups are marked with black lines (P < 0.05)
protocols for no less than 10 min were comparable with that of those without microcapsules (negative control, P > 0.05). However, regardless of the inclusion of micro- capsules or the PPT protocol, all the specimens showed the RMS values under 50 μm for overall intaglio sur- faces (Figs. 6 and 7). The color deviation map (Fig. 7) revealed that the specimens with microcapsules and subjected to 10-min PPT showed the most favorable pattern of surface deviation, with overall green area (within 50 μm of nominal deviation) with small regions of positive (yellow) or negative (blue) deviations.
Discussion
A group of researchers previously reported the fabrica- tion of denture base material with antifungal activity against C. albicans using microencapsulation of phyton- cide oil extract, mixing with a proper dispersant, and a digital light processing technique [31]. However, adding phytoncide-filled microcapsules may have affected the mechanical properties and dimensional accuracy of the 3DP denture base [37]. Although the flexural strength of the 3DP denture bases with microcapsules was com- parable with that of bases without microcapsules, the degree of conversion, hardness, or dimensional accuracy was relatively decreased [31, 37]. To improve the flexural strength, degree of conversion, hardness, and intaglio surface trueness while maintaining the antifungal effect of the 3DP denture base, the current study focused on the effect of PPT, one of the most critical phases for 3D printing. Based on the findings of this study, the null hypotheses were rejected.
Flexural strength is essential for the success of den- ture treatment, as an external loading force is exerted on the dentures during mastication [43, 44]. The flex- ural strength of 3DP denture resin was enhanced by increasing the PPT [40]. In this study, compared with 5-min PPT, 3DP denture bases with phytoncide-filled
microcapsules showed improved flexural strength after increasing the PPT to 10 min. The measured strength was higher than 65 MPa, which is necessary for base polymers to be clinically acceptable based on the recom- mendations of the International Standard Organization [41]. This may be in accordance with the significantly increased DC in the specimens with microcapsules sub- jected to 10-min PPT compared with that of those sub- jected to 5-min PPT. Generally, the pores may act as the point of crack initiation during the fracture of PMMA resin; the microcapsules incorporated in the 3DP denture base resin may also function similarly [37]. In this study, the increased PPT was beneficial in enhancing the degree of conversion and, consequently, the flexural strength.
Insufficient DC could reduce the mechanical properties of denture resins, where high DC is related to high con- version from monomers to polymers [45]. Furthermore, a denture base must have adequate hardness to resist sur- face wear during mastication or maintenance care, which may affect microbial accumulation when using complete dentures [46, 47]. The addition of microcapsules may have affected the Vickers hardness of 3DP denture base resin, still higher than the value reported in the literature [48]. In fact, by increasing PPT no less than 10 min, 3DP denture base resin with antifungal microcapsules showed sufficient Vickers hardness to withstand the clinical con- ditions [48].
For DLP-based workflow, a light source is essential as it supplies sufficient energy to induce polymerization of the photosensitive resin material, resulting in the for- mation of a highly cross-linked polymer [49]. Further- more, a post-polymerization process is required to fully polymerize the object that has just been 3DP to finalize the desired mechanical and surface properties. Among the several parameters that could be modified, PPT is one of the easiest factors to approach. By changing the PPT, the accuracy, flexural strength, and hardness of the Fig. 7 Representative images of color deviation maps of the 3D‑printed denture base resin discs subjected to different post‑polymerization time (PPT) protocols: 5‑min, 10‑min, 20‑min, and 30‑min. Control: 3D‑printed denture base resin discs without microcapsules; Capsule: 3D‑printed denture base resin discs with 5 wt% phytoncide‑filled microcapsules. a Control group, with 5‑min PPT, b Capsule group, with 5‑min PPT, c Capsule group, with 10‑min PPT, d Capsule group, with 20‑min PPT, and e Capsule group, with 30‑min PPT.
3DP prosthesis have been improved, suggesting that the increase in PPT is advantageous [38–40]. In this study, by changing PPT from 5 min (positive control) to 10 min, the antifungal activity and mechanical properties of the 3DP denture base containing microcapsules were sig- nificantly improved. However, PPT protocols longer than 10 min did not increase the antifungal effect of the 3DP denture base. In addition, the intaglio surface trueness of the 3DP denture base with microcapsules, which is para- mount for intimate tissue surface adaptation of the pros- thesis, was also evaluated in this study. With the 10-min PPT protocol, the 3DP denture bases with microcapsules showed intaglio surface trueness comparable with that of those without.
The experimental design and calculated sample size evaluated in the present study were referred to previous in-vitro research by Jeon et al. and other recent in-vitro studies with similar research designs and test materials that reported significant differences [31, 37, 40]. How- ever, the absence of a priori power analysis could be considered as a limitation of the this in-vitro research.
In addition, further in-vitro research regarding physico- mechanical properties such as fracture toughness, com- pressive strength, and color stability of 3DP denture base with microcapsules is required. Since only one of the numerous 3D-printable denture base materials was tested in this study, further experiments with other den- ture materials are required.
Conclusion
Within the limitations of this in vitro study, the 3DP den- ture base containing phytoncide-filled microcapsules at 5 wt% concentration and subjected to 10-min PPT showed sufficient antifungal activity, while maintaining clinically comparable flexural strength, Vickers hardness, and intaglio surface trueness to the original 3DP denture base resin with no microcapsules. The antifungal activity and degree of conversion of 3DP denture base resin with microcapsules were significantly affected by the change of PPT. Further in-vitro and in-vivo studies are required before applying for clinical purpose.
Acknowledgements Not applicable.
Author contributions
YHJ, WJL, and JHL carried out research and laboratory methodology. YHJ and HIY performed statistical analyses. YHJ and WJL wrote the draft. HIY edited the manuscript. HIY contributed to the study design, acquisition of the materials, interpretation of data, paper revision and supervised the research. All authors reviewed and approved the manuscript. All authors read and approved the final manuscript.
Funding
This research was supported by the Research Program to Solve Social Issues of the National Research Foundation of Korea (NRF) funded by the Ministry of
Science and ICT (NRF‑2020R1F1A1075265), the Creative‑Pioneering Research‑
ers Program through Seoul National University (SNU), and grant no. 04‑2021‑
0113 from the SNUDH Research Fund.
Availability of data and materials
The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Declarations
Ethics approval and consent to participate Not applicable.
Consent for publication Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Dental Research Institute, Seoul National University School of Dentistry, Seoul, Republic of Korea. 2 Department of Prosthodontics, School of Dentistry and Dental Research Institute, Seoul National University, 101, Daehak‑ro, Jongno‑gu, Seoul 03080, Republic of Korea.
Received: 29 July 2022 Accepted: 8 December 2022
References
1. Saeed F, Muhammad N, Khan AS, Sharif F, Rahim A, Ahmad P, Irfan M.
Prosthodontics dental materials: from conventional to unconventional.
Mater Sci Eng C Mater Biol Appl. 2020;106:110167.
2. Gundogdu M, Yanikoglu N, Bayindir F, Ciftci H. Effect of repair resin type and surface treatment on the repair strength of polyamide denture base resin. Dent Mater J. 2015;34:485–9.
3. Alkurt M, Yeşil Duymuş Z, Gundogdu M. Effect of repair resin type and surface treatment on the repair strength of heat‑polymerized denture base resin. J Prosthet Dent. 2014;111:71–8.
4. Narva KK, Lassila LV, Vallittu PK. The static strength and modulus of fiber reinforced denture base polymer. Dent Mater. 2005;21:421–8.
5. Darbar UR, Huggett R, Harrison A. Denture fracture‑A survey. Br Dent J.
1994;176:342–5.
6. Vallittu PK, Lassila VP, Lappalainen R. Evaluation of damage to removable dentures in two cities in Finland. Acta odontol scand. 1993;51:363–9.
7. Kawara M, Komiyama O, Kimoto S, Kobayashi N, Kobayashi K, Nemoto K. Distortion behavior of heat‑activated acrylic denture‑base resin in conventional and long, low‑temperature processing methods. J Dent Res. 1998;77:1446–53.
8. Nogueira SS, Ogle RE, Davis EL. Comparison of accuracy between compression‑ and injection‑molded complete dentures. J Prosthet Dent.
1999;82:291–300.
9. de Oliveira Limírio JPJ, Gomes JML, Alves Rezende MCR, Lemos CAA, Rosa CDDRD, Pellizzer EP. Mechanical properties of polymethyl meth‑
acrylate as a denture base: conventional versus CAD‑CAM resin—A systematic review and meta‑analysis of in vitro studies. J Prosthet Dent.
2021;21:S0022‑3913.
10. Javaid M, Haleem A. Current status and applications of additive manu‑
facturing in dentistry: a literature‑based review. J Oral Biol Craniofac Res.
2019;9:179–85.
11. Yoon HI, Hwang HJ, Ohkubo C, Han JS, Park EJ. Evaluation of the true‑
ness and tissue surface adaptation of CAD‑CAM mandibular denture bases manufactured using digital light processing. J Prosthet Dent.
2018;120:919–26.
12. Hwang HJ, Lee SJ, Park EJ, Yoon HI. Assessment of the trueness and tissue surface adaptation of CAD‑CAM maxillary denture bases manufactured using digital light processing. J Prosthet Dent. 2019;121:110–7.
13. Yoon SN, Oh KC, Lee SJ, Han JS, Yoon HI. Tissue surface adaptation of CAD‑CAM maxillary and mandibular complete denture bases
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manufactured by digital light processing: a clinical study. J Prosthet Dent.
2020;124:682–9.
14. Jin MC, Yoon HI, Yeo IS, Kim SH, Han JS. The effect of build angle on the tissue surface adaptation of maxillary and mandibular complete denture bases manufactured by digital light processing. J Prosthet Dent.
2020;123:473–82.
15. Da Silva WJ, Seneviratne J, Samaranayake LP, Del Bel Cury AA. Bioactiv‑
ity and architecture of Candida albicans biofilms developed on poly (methyl methacrylate) resin surface. J Biomed Mater Res B Appl Biomater.
2010;94:149–56.
16. He XY, Meurman JH, Kari K, Rautemaa R, Samaranayake LP. In vitro adhesion of Candida species to denture base materials. Mycoses.
2006;49:80–4.
17. Samaranayake LP, MacFarlane TW. An in‑vitro study of the adherence of Candida albicans to acrylic surfaces. Arch Oral Biol. 1980;25:603–9.
18. Budtz‑Jorgensen E. The role of Candida albicans in the development of stomatitis in denture wearers. Med hyg (Geneve). 1975;33:1434–5.
19. Arendorf TM, Walker DM. Denture stomatitis: a review. J Oral Rehabil.
1987;14:217–27.
20. Cumming CG, Wight C, Blackwell CL, Wray D. Denture stomatitis in the elderly. Oral Microbiol Immunol. 1990;5:82–5.
21. Gendreau L, Loewy ZG. Epidemiology and etiology of denture stomatitis.
J Prosthodont. 2011;20:251–60.
22. Webb BC, Thomas CJ, Willcox MD, Harty DW, Knox KW. Candida‑associ‑
ated denture stomatitis. Aetiology and management: a review. Part 3.
Treatment of oral candidosis. Aust Dent J. 1998;43:244–9.
23. Javed F, Samaranayake LP, Romanos GE. Treatment of oral fungal infec‑
tions using antimicrobial photodynamic therapy: a systematic review of currently available evidence. Photochem Photobiol Sci. 2014;13:726–34.
24. Emami E, Kabawat M, Rompre PH, Feine JS. Linking evidence to treat‑
ment for denture stomatitis: a meta‑analysis of randomized controlled trials. J Dent. 2014;42:99–106.
25. An S, Evans JL, Hamlet S, Love RM. Overview of incorporation of inorganic antimicrobial materials in denture base resin: a scoping review. J Prosthet Dent. 2021;27:S0022‑3913.
26. Chen SG, Yang J, Jia YG, Lu B, Ren L. TiO2 and PEEK reinforced 3D printing PMMA composite resin for dental denture base applications. Nanomateri‑
als (Basel). 2019;9:1049.
27. Monteiro DR, Gorup LF, Takamiya AS, de Camargo ER, Filho ACR, Barbosa DB. Silver distribution and release from an antimicrobial denture base resin containing silver colloidal nanoparticles. J Prosthodont.
2012;21:7–15.
28. Totu EE, Nechifor AC, Nechifor G, Aboul‑Enein HY, Cristache CM.
Poly(methyl methacrylate) with TiO2 nanoparticles inclusion for stere‑
olitographic complete denture manufacturing—the fututre in dental care for elderly edentulous patients? J Dent. 2017;59:68–77.
29. Silveira DQI, Lia EN, Massignan C, Stefani CM. Natural products for the treatment of denture stomatitis: a systematic review. J Prosthet Dent.
2021;8:S0022‑3913.
30. An S, Evans JL, Hamlet S, Love RM. Incorporation of antimicrobial agents in denture base resin: a systematic review. J Prosthet Dent.
2021;126:188–95.
31. Jeon S, Jo YH, Yoon HI, Han JS. Antifungal effect, surface roughness, and cytotoxicity of three‑dimensionally printed denture base with phyton‑
cide‑filled microcapsules: an in‑vitro study. J Dent. 2022;120:104098.
32. Salem MZM, El‑Hefny M, Ali HM, Abdel‑Megeed A, El‑Settawy AAA, Böhm M, Mansour MMA, Salem AZM. Plants‑derived bioactives: novel utilization as antimicrobial, antioxidant and phytoreducing agents for the biosyn‑
thesis of metallic nanoparticles. Microb Pathog. 2021;158:105107.
33. Abe T, Hisama M, Tanimoto S, Shibayama H, Mihara Y, Nomura M. Anti‑
oxidant effects and antimicrobial activites of phytoncide. Biocontrol Sci.
2008;13:23–7.
34. Kang SK, Auh Q, Chun YH, Hong JP. Effect of Chamaecyparis obtusa tree phytoncide on Candida albicans. J Oral Med Pain. 2010;35:19–29.
35. Lee MJ, Kim MJ, Oh SH, Kwon JS. Novel dental poly (methyl methacrylate) containing phytoncide for antifungal effect and inhibition of oral multi‑
species biofilm. Materials. 2020;13:371.
36. An S, Judge R, Wong R, Arzmi M, Palamara J, Dashper S. Incorporation of the microencapsulated antimicrobial agent phytoncide into denture base resin. Aust Dent J. 2018;63:302–11.
37. Jeon S, Jo YH, Yoon HI, Han JS. Effect of phytochemical‑filled microcap‑
sules with antifungal activity on material properties and dimensional accuracy of denture base resin for three‑dimensional printing. BMC Oral Health. 2022;22:178.
38. Lee BI, You SG, You SM, Kim DY, Kim JH. Evaluating the accuracy (true‑
ness and precision) of interim crowns manufactured using digital light processing according to post‑curing time: an in vitro study. J Adv Pros‑
thodont. 2021;13:89–99.
39. Kim D, Shim JS, Lee D, Shin SH, Nam NE, Park KH, Shim JS, Kim JE. Effects of post‑curing time on the mechanical and color properties of three‑
dimensional printed crown and bridge materials. Polymers. 2020;12:2762.
40. Aati S, Akram Z, Shrestha B, Patel J, Shih B, Shearston K, Ngo H, Fawzy A.
Effect of post‑curing light exposure time on the physico–mechanical properties and cytotoxicity of 3D‑printed denture base material. Dent Mater. 2022;38:57–67.
41. International Organization for Standardization. ISO 20795‑1: 2013. Den‑
tistry ‑ Base polymers ‑ Part 1: Denture base polymers. 2nd ed.
42. Monsen T, Lövgren E, Widerström M, Wallinder L. In vitro effect of ultra‑
sound on bacteria and suggested protocol for sonication and diagnosis of prosthetic infections. J Clin Microbiol. 2009;47:2496–501.
43. Murakami N, Wakabayashi N, Matsushima R, Kishida A, Igarashi Y. Effect of high‑pressure polymerization on mechanical properties of PMMA denture base resin. J Mech Behav Biomed Mater. 2013;20:98–104.
44. Kawaguchi T, Lassila LV, Sasaki H, Takahashi Y, Vallittu PK. Effect of heat treatment of polymethyl methacrylate powder on mechanical properties of denture base resin. J Mech Behav Biomed Mater. 2014;39:73–8.
45. Franz A, König F, Lucas T, Watts DC, Schedle A. Cytotoxic effects of dental bonding substances as a function of degree of conversion. Dent Mater.
2009;25:232–9.
46. Cevik P, Yildirim‑Bicer AZ. The effect of silica and prepolymer nano‑
particles on the mechanical properties of denture base acrylic resin. J Prosthodont. 2018;27:763–70.
47. Machado AL, Breeding LC, Vergani CE, da Cruz Perez LE. Hardness and surface roughness of reline and denture base acrylic resins after repeated disinfection procedures. J Prosthet Dent. 2009;102:115–22.
48. Al‑Dwairi ZN, Al Haj Ebrahim AA, Baba NZ. A comparison of the surface and mechanical properties of 3D printable denture‑base resin material and conventional polymethylmethacrylate (PMMA). J Prosthodont. 2022.
Epub ahead of print.
49. Zakeri S, Vippola M, Levänen E. A comprehensive review of the photopo‑
lymerization of ceramic resins used in stereolithography. Addit Manuf.
2020;35:101177.
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